Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
  • C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase

Definitions

• This invention relates to certain fluorescent substrates and a method for detecting organophosphatase activity in general, and paraoxonase activity specifically and in particular in biological fluids such as blood and serum, through the use of such fluorescent substrates.
• Enzymatic degradation of organophosphates is performed by specialized enzymes including bacterial organophosphorus hydrolase (OPH) and mammalian paraoxonase.
• OHP organophosphorus hydrolase
• Paraoxonase also referred to as, arylesterase (EC 3.1.1.2) is a 43 kDa molecular weight calcium dependent ester hydrolase that catalyses the hydrolysis of a broad range of esters such as OPs, and unsaturated aliphatic and aromatic carboxylic esters. Its name derives from the ability of this protein to hydrolyze paraoxon, the toxic metabolite of the insecticide parathion.
• paraoxonase In addition to paraoxon, paraoxonase is able to detoxify a number of other insecticides, e.g. diazonin, as well as the potent nerve gases sarin and soman that target acetylcholinesterase (AChE).
• the paraoxonase gene (PON) family consists of at least three members: PON1, PON2 and PON3, which are located on the human 7q21.3-22.1 chromosome. No significant endogenous expression of PON2 and PON3 genes has been detected. Most PON1 expression takes place in the human liver; from there the protein is secreted into blood where it circulates associated with high density lipoprotein (HDL) particles.
• HDL high density lipoprotein
• Paraoxonase has the unusual property that the mature protein retains its hydrophobic N-terminal signal peptide, which is used as an anchor for association with HDL.
• the enzyme has three potential N-linked sites and carbohydrate accounts for approximately 16% of its molecular mass.
• LDL low density lipoprotein
• Paraoxonase has been shown both to prevent formation of oxidized LDL and to hydrolyze LDL-derived oxidized phospholipids. Since accumulation of oxidized LDL is one of the key factors in development of atherosclerosis, paraoxonase activity may correlate with development of this disease. For example, Shih et al demonstrated that PONl -/- mice were extremely sensitive to diet-induced atherosclerosis in comparison with wild type mice.
• LPS lipopolysaccharide
• HDL high density lipoprotein
• PON-1 knockout mice are extremely sensitive to LPS [6].
• Paraoxonase is able to hydrolyze a number of OP toxins in vitro, and the ability of paraoxonase to protect animals in acute OP poisoning has been extensively studied. Injection of purified paraoxonase protected animals against OP toxicity [7, 8]. Further proof of the ability of paraoxonase to protect animals has been obtained from studies on PONl "knock-out" mice. Destruction of the PONl gene by knock-out technology creates mice that lack paraoxonase. Compared to wild type littermates, PONl deficient mice were extremely sensitive to the toxic effects of chlorpyrifos, an OP.
• monitoring of paraoxonase activity may help to evaluate a person's ability to withstand OP poisoning associated with deployment of chemical weapons.
• monitoring blood levels of paraoxonase may be used to identify, a predisposition to atherosclerosis, sepsis and OP poisoning.
• the absence of a robust test for detection of paraoxonase levels in blood has significantly delayed progress in studying the diagnostic value of paraoxonase.
• There are two major options for detection of this enzyme activity The first is a change in optical density and the second the generation of a fluorescent product.
• This reaction has a much higher Vmax, than the Vmax of paraoxon hydrolysis; however, phenylacetate is also hydrolyzed by a number of other esterases in cell extracts and serum samples, which significantly decreases the specificity of detection.
• the detection of phenylacetate hydrolysis is based on monitoring adsorption at 270 nm making paraoxonase detection difficult, or impossible, in protein rich solutions or in extracts containing detergents like Triton X-100.
• Pseudomonas diminuta and Flavobacterium sp It has been suggested that this enzyme evolved recently in these bacteria in response to industrial soil contamination with organophosphate compounds. Like paraoxonase, OPH catalyzes a broad range of organophosphate esters including sarin and VX. Due to this activity these organisms may have additional utility in decontamination of OPs in the environment. In this context a sensitive and robust assay would be necessary to confirm expression of OPH in the presence of a large excess of phosphatase activity. Thus, it is essential that the substrate has very little or no affinity for phosphatases.
• the present invention provides highly sensitive and specific fluorescent substrates.
• the present invention provides compounds of the formula (I):
• R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo or hydrogen; X 1 , X 2 , and X 3 are independently O or S; provided that R 9 and R 10 are not simultaneously H, when all of X 1 , X 2 , and X 3 are
• R ⁇ -R 14 are selected from the group consisting of H and groups or atoms other than H; X 4 - X 9 are independently O or S. n and m are 0 or 1 but m and n cannot be 0 simultaneously. R 15 - R 24 can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent compound.
• the present invention also provides a method for detecting and/or measuring the organophosphatases and particularly paraoxonase activity in a fluid comprising contacting the fluid with a fluorescent substrate and measuring the fluorescence of the fluorescent product formed.
• the fluorescent substrates of this invention are specific for organophosphatases including paraoxonase and, when hydrolyzed, release highly fluorescent products which can be measured at, for example, an emission wavelength of 460 nm following excitation at a wavelength of 355 nm for structures based on the coumarin structure and emission of 520 nm following excitation at 488 nm for fluorescein-based structures.
• an emission wavelength of 460 nm following excitation at a wavelength of 355 nm for structures based on the coumarin structure and emission of 520 nm following excitation at 488 nm for fluorescein-based structures.
• these have significantly higher sensitivity and specificity.
• the substrates of the present invention facilitate large throughput methods for the detection and quantitation of this enzyme's activity.
• the substrates are useful for detecting and quantifying paraoxonase activity in samples of biological fluids such as blood. Measurement of blood paraoxonase activity may be useful as an indicator of cardiovascular disease and sensitivity to OP poisoning. Also provided is a method for detecting the activity of paraoxonase in an environmental sample. Such samples may include those which have been treated with paraoxonase to decontaminate OPs. Also provided is a method for studying the basic properties of paraoxonase by using these substrates as research reagents.
• the substrates of the present invention have one or more than one advantage; e.g., high specificity for paraoxonase; high sensitivity fluorescent detection and a significant Vmax of reaction makes it at least 10-20 times more sensitive than any other known substrate for paraoxonase detection. Consequently, the substrates may have a significant practical use in different areas of medicine and detection of nerve gas poisons.
• the substrates are useful for detecting and quantifying OPH activity in environmental samples such as soil extracts or swabs. Such samples may include those which have been treated with OPH to decontaminate OPs.
• the substrates of the present invention have one or more than one advantage; e.g., high specificity for OPH; high sensitivity fluorescent detection and a significant Vmax of reaction makes it at least 10-20 times more sensitive than any other known substrate for OPH detection. Consequently, the substrates may have a significant practical use in different areas of medicine and detection of nerve gas poisons.
• the proposed substrates can be used for broad screening of paraoxonase activity in human blood.
• the present invention provides an assay kit for paraoxonase detection and quantitation.
• a kit may be used for detection of paraoxonase as a diagnostic marker for prediction of atherosclerosis development.
• the diagnostic prognosis of paraoxonase detection is comparable to, or better than, such blood markers as blood cholesterol level.
• Such a kit may also be used for detection of paraoxonase as a diagnostic marker for prediction of sepsis development.
• kits for detection of paraoxonase are predicting the resistance to OP challenges which can have a significant value in a war against chemical terrorism or during combat where chemical weapons are utilized. It is also envisaged that the kit may be used to confirm that protective levels of paraoxonase have been achieved in war fighters following administration of prophylactic levels of recombinant paraoxonase.
• a rapid and sensitive method for detection of organophosphatase activity may be useful for the detection of alternative substrates, e.g., nerve poisons, OP toxins and insecticides, present in environment samples.
• Such alternative substrates for organophosphatase may be identified by their ability to compete for binding and hydrolysis of the substrates of the present invention.
• the present invention further provides a method for selectively detecting organophosphatase in a sample suspected to contain organophosphatase and a phosphatase comprising contacting the sample with a substrate of the invention, measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the organophosphatase enzyme.
• the spectrum of structures provides a method to discover different organophosphatases with different spectra of substrate specificities.
• the present invention further provides a method for detecting and/or measuring the activity of organophosphatase enzyme immobilized on a support comprising contacting the support with a substrate of the invention, measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the organophosphatase enzyme.
• Figure 1 depicts the detection of paraoxonase activity in serum samples using
• Figure 2 depicts a cell membrane associated production of fluorescence in
• Figure 3 depicts the measurement of the Km of DEPFMU for rabbit recombinant paraoxonase.
• the rabbit recombinant paraoxonase was expressed in CHO cells and partially purified.
• v-A, v-B and v-C are the velocities at 1/5, 1/10 and 1/20 dilutions of recombinant paraoxonase, respectively.
• Partially purified recombinant paraoxonase was mixed with solutions containing different concentrations of DEPFMU as substrate. The time-course of DEPFMU hydrolysis 37°C was monitored using fluorescence reading at
• Figure 4 depicts a comparison of the sensitivity of paraoxonase detection using paraoxon and DEPFMU based assays. An evaluation of the relative sensitivity of
• DEPFMU (A) and paraoxon (B) based assays for paraoxonase assay was performed using
• Figure 5 depicts the hydrolysis of the DEPFMU by PONl mutants.
• Figure 6 A depicts the hydrolysis data for a substrate of the present invention
• FIG. 6B depicts the hydrolysis data for 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). See Example 8.
• Figure 7 depicts the hydrolysis of the fluorescein diphosphate terra ethyl ester (FDPTEE) by bacterial OPH.
• Figure 8 depicts the hydrolysis of FDPTEE by normal mice contrasted with
• the present invention provides a compound of the formula I:
• R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and , and X are independently O or S; provided that R 9 and R 10 are not simultaneously H, when all of X 1 , X 2 , and X 3 are O.
• Ci-C 6 aminoalkyl C 5 -C 8 cycloalkyl, Ci-C ⁇ haloalkyl, C 5 -C 8 halocycloalkyl, Ci-C ⁇ hydroxyalkyl, C 5 -C 8 hydroxycycloalkyl, Ci- C 6 alkoxy Ci-C ⁇ alkyl, C 2 -C 6 alkoxycarbonyl, C 2 -C 6 alkoxycarbonyl Ci-C ⁇ alkyl, carboxy Ci-C ⁇ alkyl, carboxy Ci- C 6 alkoxy, dicarboxy Ci-C ⁇ alkyl, dicarboxy C C 6 alkoxy, C 2 -C 6 cyanoalkyl, phosphono Ci-C ⁇ alkyl, phosphoryl C!-C 6 alkyl, mono-, di-, and trisaccharides, nucleic acids, oligonucleotides, amino acids, peptides, and proteins, and C 2 -C 6 alkenyl, C 2 - C
• R 4 is selected from the group consisting of H, hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, sulfomethyl, salt of sulfomethyl, thiocyanato, aldehydo, keto, carbamoyl, urethane, ureido, guanidino, Ci-C ⁇ alkylamino, Ci-C ⁇ acylamino, Ci-C ⁇ alkylamido, Ci-C ⁇ alkyl, C C ⁇ alkoxy, C C ⁇ perfluoroalkyl, halomethyl, CrC ⁇ alkylthio, C 5 -C 8 cycloalkyl, - haloalkyl, C 5 -C 8 halocycloalkyl, Ci-C ⁇ hydroxy
• R 4 is selected from the group consisting of H, cyano, sulfomethyl, salt of sulfomethyl, aryl, Ci-C ⁇ alkyl, Ci-C ⁇ alkoxy, and Ci-C ⁇ perfluoroalkyl, more preferably -C ⁇ alkyl, for example, methyl.
• R 9 and R 10 are selected from the group consisting of H, Ci-C ⁇ alkyl, C 5 -C 8 cycloalkyl, C ⁇ -C 6 haloalkyl, Ci-C ⁇ perfluoroalkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl, and aryl, arylcarbonyl, and heteroaryl, which may be optionally substituted with a substituent selected from the group consisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydo, keto, carbamoyl, urethane, ureido, and guanidino; and X 1 , X 2 , and X 3 are O or
• R 9 and R 10 are selected from the group consisting of H, - alkyl, C -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, and heteroaryl, more preferably from the group consisting of H, Ci-C ⁇ alkyl, C -C 6 alkenyl, and C 2 -C 6 alkynyl.
• R 9 and R 10 are selected from the group consisting of Ci-C 6 alkyl, for example, R 9 and R 10 are ethyl.
• R 5 is H or Ci-C ⁇ alkoxy, preferably H.
• R and R are fluoro. Examples of preferred compounds include those wherein X 1 , X 2 , and X 3 are O or S, more preferably O, R 9 and R 10 are ethyl, R 4 is methyl, R 6 and R 8 are fluoro, and R 3 and R 5 are H.
• the compound of formula I are those wherein R 9 and R 10 are ethyl, R 4 is methyl, R 6 and R 8 are fluoro, and X 1 , X 2 , and X 3 are O; and those wherein X 1 and X 2 are O, X 3 is S, R 6 and R 8 are H; R 9 and R 10 are ethyl, and R 4 is methyl.
• the compounds of the present invention can be prepared by any suitable method, for example, by following methods generally known in the art; see, e.g., U.S. Patents 4,659,657; 5,428,059; 5,830,912; and 6,416,970; and U.S. patent application publication No.
• R u -R 14 are selected from the group consisting of H and groups or atoms other than H; X 4 - X 9 are independently O or S. m and n are 0 or 1 but m and n cannot be 0 simultaneously. R 15 - R 24 can be H or any substituent so long as the compound of formula II upon hydrolysis, e.g., of the P-X 6 and/or P-X 9 bonds, provides a fluorescent compound. When m or n is 0, the substituent at that position is H.
• R 11 - R 14 are independently selected from the group consisting of H, -C ⁇ alkyl, C 5 -C 8 cycloalkyl, C!-C 6 haloalkyl, Ci-C 6 perfluoroalkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl, and aryl, arylcarbonyl, and heteroaryl, which may be optionally substituted with a substituent selected from the group consisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydo, keto, carbamoyl, urethane, ureido, and guanidino; and X 4 - X 9 are independently O or S, preferably O.
• R 15 - R 24 are independently selected from the group consisting of H, hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, sulfomethyl, a salt of sulfomethyl, thiocyanato, aldehydo, keto, carbamoyl, urethane, ureido, guanidino, C C 6 alkylamino, Ci-C ⁇ acylamino, Ci-C ⁇ alkylamido, Ci-C ⁇ alkyl, C ⁇ -C 6 alkoxy, Ci-C ⁇ alkylthio, C 5 -Cs cycloalkyl, d-Ce haloalkyl, -Ce perfluoroalkyl,
• R 11 - R 1 are independently selected from the group consisting of H, -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, and heteroaryl, more preferably from the group consisting of H, Ci-C ⁇ alkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl.
• R 11 - R 14 are independently selected from the group consisting of Ci-C ⁇ alkyl, for example, R 11 - R 1 are ethyl, and m and n are 1.
• X 4 -X 9 are O
• R 15 -R 24 are H
• R ⁇ -R 14 are ethyl
• m and n are 1
• X 4 , X 5 , X 7 , and X 8 are O
• X 6 and X 9 are S
• R 15 -R 24 are H
• R u -R 14 are ethyl
• m and n are 1.
• aryl is a 1-3 aromatic ring containing group, preferably phenyl; heteroaryl is a 5- or 6- membered aromatic heterocycle that is optionally fused to an additional 6-membered aromatic ring or to one or more heteroaromatic ring containing 1-3 heteroatoms selected from the group consisting of O, N, and S.
• heteroaryl include pyrrole, thiophene, furan, oxazole, isooxazole, or imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, and indole.
• the compounds of formula II can be prepared by any suitable method.
• fluorescein diphosphate tetraethyl ester can be prepared as shown in Example 10.
• the compounds of the present invention can be linked or conjugated to other molecules, groups, or substance, e.g., a dye, a reactive group, an antibody, or a solid support.
• “Assay Buffer” is the buffer used in the detection of paraoxonase activity and is composed of 20 mM Tris.HCl, 150 mM NaCI and 2 mM CaCl 2 at pH 8.0 at 25 °C.
• Bio fluid is a sample of a fluid originating from a biological source.
• biological fluids include, but are not limited to blood, blood-derived compositions, serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, semen, cell or tissue extracts, culture medium from the expression of paraoxonase or mutations of paraoxonase, samples arising from the fractionation of paraoxonase or HDL from biological samples.
• DEPFMU is the abbreviation for 7-diethyl-phospho-6,8-difluoro-4- methylumbelliferyl, a chemical compound that is one of the newly invented fluorogenic substrates for detection of paraoxonase activity
• Environmental sample is a sample obtained from the environment for purposes of detection of paraoxonase or OPs. It may be soil, water, air or any other material obtained natural environment.
• FDPTEE fiuorescein diphosphate tetraethyl ester
• organophosphate is the abbreviation for organophosphate, which includes a variety of organic compounds that contain phosphorus and often have intense neurotoxic activities.
• OPH refers to the protein encoded by organophosphorus hydrolase which is expressed by two soil dwelling bacteria, Pseudomonas diminuta and Flavobacterium sp. It hydrolyses a number of organic esters including paraoxon.
• Paraoxonase refers to the protein encoded by PONl gene. Paraoxonase is a serum protein that possesses enzymatic activity. It hydrolyzes a number of organic and phospho-organic esters including paraoxon. The physiological function of paraoxonase is not known with certainty.
• PONl refers to the gene encoding the protein known as paraoxonase, or arylesterase (EC 3.1.1.2). Paraoxonase is present in normal human plasma and the cDNA, and genes encoding the human protein have been sequenced and characterized.
• Substrate for organophosphatase refers to one of a number of chemical compounds that are hydrolyzed by OPH and/or paraoxonase. These include DEPFMU, phenyl acetate, oxidized lipids and paraoxon.
• 355/460 refers to an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
• the present invention further provides a method for detecting and/or measuring the activity of organosphosphatase in a fluid comprising contacting the fluid with a compound of formula I, wherein R 3 -R 6 and R 8 -R 10 can be any atom or group and X 1 , X 2 , and X 3 are independently O or S; or of the formula II, wherein R ⁇ -R 14 are selected from the group consisting of H and groups or atoms other than H; X 4 - X 9 are independently O or S. n and m are 0 or 1 but m and n cannot be 0 simultaneously.
• R 15 - R 24 can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent compound; measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the paraoxonase enzyme. Any of the compounds described above in paragraphs [0029] -[0036] and [0038] - [0042] may be employed in the method of the present invention.
• the compound of formula I can be one wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo.
• the compound of formula II can be one wherein X 4 -X 9 are O, m and n are 1, R n -R 24 are H.
• the compounds described above can be used for detection of organophosphatase activity and specifically paraoxonase activity in a biological fluid.
• biological fluids include, but are not limited to, blood, blood- derived compositions or serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, semen, brain, artery, vein and gland extracts.
• Other fluids may contain culture medium from the expression of paraoxonase or mutations of paraoxonase.
• Still further fluids may be taken from methods and processes resulting in the fractionation of paraoxonase or HDL from biological samples.
• the fluid is an environmental fluid, for example, an extract of soil, water, or swab.
• the compounds described above can be used for detection of organophosphatase activity and specifically OPH activity in a biological fluid or environmental extract.
• biological fluids include, but are not limited to, blood, blood-derived compositions or serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, semen, brain, artery, vein and gland extracts.
• Other fluids may contain culture medium from the expression of OPH or mutations of OPH.
• Still further fluids may be taken from methods and processes resulting in the fractionation of OPH from biological samples.
• the fluid is an environmental fluid, for example, an extract of soil, water, or swab.
• the present invention provides a method for predicting the existence of cardiovascular diseases.
• the present invention further provides a method for predicting a person's sensitivity to OPs.
• the activity of paraoxonase as measured using the compounds of the present invention can be used as a predictor of cardiovascular disease and sensitivity to OPs.
• the present invention provides a method for predicting the potential for septic shock.
• the present invention further provides a method for predicting a person's sensitivity to LPS.
• the activity of paraoxonase as measured using the compounds of the present invention can be used as a predictor of sensitivity to LPS.
• the present invention provides a method for evaluating and/or predicting the functional activity of preparations of HDL.
• the paraoxonase activity measured by the fluorescence in accordance with the present invention may be used for evaluation/prediction of functional activity of preparations of high density lipoproteins.
• detection of fluorescence is achieved using an excitation wavelength and an emission wavelength.
• OPH and paraoxonase activity can be monitored using a fluorimeter with an excitation wavelength at 355 nm and an emission wavelength at 460 nm.
• Other assay formats include analysis of OPH activity in gels after protein separation by electrophoresis or analysis of paraoxonase expression/secretion in live or dead cells embedded in low melting point agarose, immunoblotting, western blot analysis, and fluorescent detection in situ with detection by microscopy, visual inspection, via film.
• OPH or paraoxonase may be immobilized on supports such as membranes, resins or dipsticks.
• organophosphatase activity may be detected on the surface of cells expressing paraoxonase activity using a cell sorter (e.g., fluorescence assisted cell sorter or FACS).
• a cell sorter e.g., fluorescence assisted cell sorter or FACS.
• the compounds of the present invention can be used to quantify the activity of OPases such as those associated with paraoxonase or variants of paraoxonase including natural variants or artificially created mutant forms of paraoxonase.
• OPases such as those associated with paraoxonase or variants of paraoxonase including natural variants or artificially created mutant forms of paraoxonase.
• organophosphatases such as those associated with OPH or variants of OPH including natural variants or artificially created mutant forms of OPH.
• the presence of competing substrates (or compounds) for organophosphatase can be identified by inhibition of fluorescence in the presence of a substrate for OPH or paraoxonase.
• the substrate is DEPFMU and the competing substrate is an OP such as sarin or soman.
• paraoxonase is immobilized on a support and its activity is measured by the production of a fluorescent signal.
• Environmental extracts including water and air, extracts of swabs are added to paraoxonase and alternative substrates identified by a decrease in fluorescence.
• Environmental extracts may be extracted with aqueous or organic solvents, supercritical fluids, subcritical fluids, and the like.
• OPH is immobilized on a support and its activity is measured by the production of a fluorescent signal.
• Environmental extracts including water and air, extracts of swabs are added to OPH and alternative substrates identified by a decrease in fluorescence.
• Environmental extracts may be extracted with aqueous or organic solvents, supercritical fluids, subcritical fluids, and the like.
• organic and inorganic polymers may be employed as the material for immobilizing organophosphatases such as
• Illustrative polymers include polyethylene, polypropylene, polymethacrylate, polyacrylate, rayon, nylon, cellulose, nitrocellulose, and polyvinylidene fluoride.
• the immobilized organophosphatase can be quantified by contacting with a compound of the present invention.
• the present invention provides a method for monitoring decontamination of the environment of OPs.
• the extract of the soil treated with paraoxonase (for decontamination) can be contacted with a compound of the present invention, and the fluorescence produced can be an indication of the completeness of decontamination.
• the compound of the present invention can be employed to identify soil-dwelling micro-organisms or plants which express either OPH and/or paraoxonase.
• the organophosphatase is coupled to a secondary structure such as an antibody with specificity for an alternative target such that the secondary structure binds to its target to form a organophosphatase-secondary structure: target complex.
• the organophosphatase may then be used as a reporter protein and the presence of the organophosphatase-secondary structure: target complex identified using the substrates or compounds of the present invention.
• the PONl gene is co-transfected with another protein of interest and used as a reporter gene for expression of the target protein.
• the PONl gene is under the control of different promoters and the fluorescent substrate used to determine the activity of different promoters.
• the OPH gene is co-transfected with another protein of interest and used as a reporter gene for expression of the target protein.
• the OPH gene is under the control of different promoters and the fluorescent substrate used to determine the activity of different promoters.
• the substrate is added to cells incubated with different molecules that may up-regulate the PONl promoter and hence the expression of paraoxonase.
• Up-regulators of paraoxonase expression are identified by the increase in fluorescent signal in the presence of paraoxonase substrate. These regulators may affect signal transduction pathways that ultimately result in up-regulation of the gene promoter.
• DEPFMU may be used as a paraoxonase specific substrate which can accurately detect serum paraoxonase activity even in the presence endogenous phosphatase. Consequently, a major advantage of DEPFMU over prior art, is the unexpectedly low specificity of this substrate to phosphatase and its high specificity for organophosphatases such as OPH and paraoxonase.
• the present invention further provides a method for detecting and/or measuring the activity of organophosphatase including paraoxonase immobilized on a support comprising contacting the support with any of the compounds of formula I or II; measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the paraoxonase enzyme.
• the support is a membrane, resin, biosensor, microtiter plate, nanotube or dipstick, fiber, silicon chip, magnetic beads, and different gels.
• the present invention further provides a method for selectively detecting organophosphatase in a sample suspected to contain organophosphatase and a phosphatase comprising contacting the sample with any of the compounds of formula I or II , e.g., the compound of formula I, wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo or H; and the compound of formula II, wherein X 4 -X 9 are O, m and n are 1, R ⁇ -R 2 are H; measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the organophosphatase enzyme.
• the compounds of formula I or II e.g., the compound of formula I, wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other
• the present invention further provides a method for detecting and/or measuring the activity of organophosphatase enzyme immobilized on a support comprising contacting the support with any of the compounds of formula I or II; e.g., the compound of formula I, wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo or H; and the compound of formula II, wherein X 4 -X 9 are O, m and n are 1, R ⁇ -R 24 are H; measuring the fluorescence of a fluorescent product formed during the contacting; and correlating the measured fluorescence with the activity of the organophosphatase enzyme.
• the compound of formula I wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo or H
• the compound of formula II where
• the substrates of the present invention provide unexpected specificity and sensitivity for detection of OPases even in the presence of the high acid and alkaline phosphatase activities found in cellular extracts, plasma and sera.
• the fact that the substrates of the present invention are even recognized by organophosphatases is surprising in view of the large size of the fluorogenic groups used relative to the known substrates for these proteins.
• organophosphatases such as paraoxonase let alone selectively hydrolyzed by the protein.
• the tested compounds included: 4-methylumbelliferyl acetate, 4- methylumbelliferyl oleate, 4-methylumbelliferyl heptanoate, 4-methylumbelliferyl palmitate, 6,8-difluoro-4-methylumbelliferyl-octanoate, 7-[diethyl-phospho]-6,8-difluoro-4- methylumbelliferyl [DEPFMU], and ELF-97 palmitate (Molecular Probes, OR), fluorescein diphosphate tetraethyl ester, (3-carboxypropyl)-trimethylammonium chloride 4- methylumbelliferyl ester, and 7-benzyloxy-6,8-difluoro-4-methylumbelliferyl.
• Amplified PCR product containing PONl cDNA was cloned into pBlueScript KS II vector (Stratagene, CA) as Xbal/ EcoRI fragment, sequenced from T3 and T7 primers to confirm identity of DNA and then subcloned into an expression vector containing EF-la promoter and GC-MSF poly (A) signal, forming expression vector pHLSS 131.
• the expression vector DNA was propagated in E. coli, DH5a strain, and purified using Quagen Maxiprep kit for plasmid purification (Quagen, CA). CHO cells were obtained from ATCC and cultivated according to the recommended conditions.
• CHO cells were transfected with the expression vector using Fugene 6 reagent (Roche Diagnostic, Indianapolis, IN) according the manufacturer's manual. 48 hours after transfection, the level of PONl expression was easily detectable by using standard substrates like paraoxon and phenylacetate.
• the test compounds were screened for paraoxonase mediated hydrolysis by incubation with transfected CHO cells. Fluorescent monitoring of the reaction was performed using SpectraMax GenimiXS fluorimeter, Molecular Devises Inc. (Sunnyvale, CA).
• DEPFMU was specifically hydrolyzed by paraoxonase. After hydrolysis, highly fluorescent 6,8-difluoro-4-methylumbelliferyl is released. Since the DEPFMU itself does not possess any significant fluorescence, hydrolysis can be easily and safely monitored using any commercial fluorimeter with excitation at 355 nm and emission at 460 nm.
• EXAMPLE 2 [0083] This example illustrates an assay for detection of paraoxonase activity.
• DEPFMU may be used as a substrate for detection of serum derived as well as recombinant paraoxonase expressed in cell cultures. The following conditions have been used for the detection of paraoxonase in plasma.
• Samples of serum or plasma may be diluted 1 to 100 times in an assay buffer. This assay was performed in a 96 well "Nunc-immuno" plate. For example, 10 ⁇ l of diluted rabbit serum was added to each well of a 96 well plate containing 100 ⁇ l of assay buffer plus 100 ⁇ M of DEPFMU (stock DEPFMU was prepared as a 50 mM concentrate in dimethylformamide and stored at -20°C). After thorough mixing the assay solution containing plasma was incubated for 20 min.
• EXAMPLE 3 This example illustrates a method of detecting recombinant paraoxonase activity in CHO cells transfected with paraoxonase.
• Human PONl cDNA was recovered, cloned into pBlueScript KS II, and sequenced to confirm identity.
• cDNA was subcloned in expression vector under the control of the EF-la promoter.
• CHO cells were transfected using Fugene 6 reagent with expression vector pHLSS122, containing human PONl cDNA under EF-la promoter in pBlueScript KS II cloning vector. Transfection with pBlueScript KS II vector DNA was performed for control CHO cells. Transfected cells were plated into 96 well tissue culture plates.
• EXAMPLE 4 This example illustrates a method for measuring the Km of recombinant rabbit paraoxonase for DEPFMU.
• CHO cells were transfected with the plasmid pHLS S 131 expressing rabbit
• PONl cDNA See example 1 and 2 for additional details of transfection and expression. After transfection the cells were propagated in growth medium, harvested and paraoxonase was partially purified from the cell membrane by extraction with 0.03% Tergitol (Sigma) in PBS. Extracted paraoxonase was separated from cells by centrifugation at lOOOOg for 10 min. The supernatant was considered as partially purified paraoxonase.
• This example provides a comparison of the sensitivity of paraoxon and a compound of the present invention as a substrate for paraoxonase detection.
• This experiment demonstrates the relative sensitivity of DEPFMU-based assay compared with paraoxon based assays. 10 ⁇ l of serially diluted samples of rabbit serum were incubated for 30 min at 37°C in the presence of 100 ⁇ l of 4 mM paraoxon or 100 ⁇ l of 100 ⁇ M DEPFMU in the assay buffer described above. After incubation, the optical density change of paraoxon was measured at a wavelength of 405 nm and the fluorescence of DEPFMU hydrolysis was measured at 355/460. The data is presented in Figure 4.
• EXAMPLE 6 This example illustrates the hydrolysis of DEPFMU and detection of PONl activity in CHO cells transfected with different PONl mutants.
• DNA vector contained PONl cDNA driven by EF-1 promoter. Since different natural variants of PONl have different substrate specificity, four different variant/mutants of PON were used (145-6, 142-2, 131-10, and 122-7). 48 hours after transfection, cells were washed with PBS and 100 ⁇ L of the DEPFMU substrate were added. Final concentration of the substrate was 40 ⁇ g/mL in 50 mM Tris buffer pH 8.0, 100 mM NaCI, and 1 mM CaCl 2 . After 10 min. of incubation at 37C, fluorescence was measured at 355 nm excitation and 460 nm emission. The results are shown in Figure 5. All four mutants hydrolyzed the DEPFMU with very high efficiency. Spontaneous hydrolysis of substrate by CHO non-transfected cells was about 3%. The control CHO cell did not hydrolyze the substrate.
• EXAMPLE 7 This example illustrates the sensitivity of detection provided by an embodiment of the present invention.
• a serial dilution of OPH from 10 ⁇ g /ml to 0.5 ng/ml using different substrates including paraoxon and DEPFMU.
• 10 ⁇ l of OPH solution was mixed with 100 ⁇ l of buffer containing 100 ⁇ M of DEPFMU, or 1.2 mM of paraoxon. Samples were incubated for 30 min at 37 °C with changes in optical properties monitored every 5 min.
• the k cat for paraoxon was more than 10 times higher than the kca t f° r DEPFMU, the superior signal to noise ratio for the coumarin fluorophore over the optical change in nitrophenol, which was released after paraoxon hydrolysis, makes the DEPFMU based assay system approximately 100 times more sensitive for OPH detection than paraoxon.
• the Km of DEPFMU for OPH was analyzed. For this experiment 10 ⁇ l of OPH solution containing 25 fmoles of enzyme were mixed with 100 ⁇ l of 100 ⁇ M DEPFMU. The velocity of hydrolysis was constant only during the first 10 min of reaction and decreases after 10-15 min.
• the Km of DEPFMU for OPH was evaluated using concentrations of DEPFMU ranging from 290 ⁇ M to 2 ⁇ M. 10 ⁇ l of solution containing 25 fmoles of OPH were mixed with 100 ⁇ l of substrate solution. The reciprocal of the Km of DEPFMU was obtained from the intercept on the abscissa using a Lineweaver-Burk Plot (1/v) where v is the velocity of the reaction against 1/[S] where [S] is the substrate concentration. The apparent Km was calculated as 29 ⁇ M.
• EXAMPLE 8 This example illustrates a superior property of a substrate of the present invention, DEPFMU, relative to that of 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as substrate for detection of paraoxonase.
• CHO cells were transfected using Fugene 6 reagent with the expression vector pHLSS122, containing human PONl cDNA under EF-la promoter in pBlueScript KS II cloning vector. Transfection with pBlueScript KS II vector DNA was performed for control CHO cells.
• Different plasmids 182,178,190,184 188 and 186 containing different mutants and natural variants of PONl were used for expression.
• Transfected cells were plated into 96 well tissue culture plates. 48 hours after transfection, the wells were washed twice with PBS and 100 ⁇ l of assay buffer containing 100 ⁇ M DEPFMU or 100 ⁇ M DiFMUP was added. Immediately after addition, the fluorescence of the wells was monitored using a 96 well SpectraMax Gemini XS fluorescence reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The data from this experiment is presented on Figure 6a and 6b. On Figure 6a, DEPFMU hydrolysis by control and transfected cells was demonstrated.
• FIG. 6b demonstrates data on DiFMUP hydrolysis by control and transfected CHO cells.
• the control cells as well as experimentally transfected cells do hydrolyze significant amount of DiFMUP, indicating there is significant PONl independent hydrolysis of DiFMUP. Most probably this high level of hydrolysis is mediated by cell phosphatases, which are abundant in the majority of cell lines.
• the significant paraoxonase independent hydrolysis of DiFMUP precludes this substrate being useful for the detection of paraoxonase.
• EXAMPLE 9 This example illustrates a method for the detection of organophosphatase.
• Fluorescein diphosphate tetraethyl ester (FDPTEE) was used as a substrate for detection of organophosphatase activity. The experiment was performed as described above in example 7. Briefly, serial dilution of OPH from 10 ⁇ g /ml to 0.5 ng/ml was prepared. 10 ⁇ l of the various dilution of OPH were mixed with 100 ⁇ l of buffer containing 100 ⁇ M of FDPTEE. Samples were incubated for 30 min at 37 °C and fluorescence (Ex 488 nm/ Em 520 nm) was monitored every 5 min. The results obtained are shown in Figure 7.
• the enzyme has a molecular weight of 39,000 kDa and is 100% pure and fully active as low as 100 femtomole (fmol) of OPH per well was reliably detected, showing it to be applicable as a substrate for OPase.
• EXAMPLE 10 This example illustrates a method of preparing an embodiment of the compound of formula II, namely, fluorescein diphosphate tetraethyl ester.
• 2.5 g of fluorescein (7.5 mmol) is suspended in THF (60 mL) and anhydrous CH 2 CI 2 (mL).
• 3.1 g of lH-tetrazole 3.1 g.(44 mmol) is added and stirred at room temperature for ⁇ 1.5 hours or until the reaction mixture becomes a transparent dark yellowish solution.
• R f fluorescein diphosphite, tetraethyl ester
• R f fluorescein diphosphite, tetraethyl ester
• MCPBA 3- chloroperoxybenzoic acid
• Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different fi'om that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q andR. Arterioscler Thromb Vase Biol 18, 1617-24.

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